M csf

In vitro osteoclastogenesis assays were performed as described previously^{18 (link),112 (link)} with modifications. Briefly, mouse bone marrow cells were harvested from 4-week-old male C57BL/6J mice by flushing marrow cavity from both tibia and femur, filtered through 40 µm cell strainer, and cultured with alpha minimum essential medium (α-MEM) containing 15% fetal bovine serum (FBS), 100 U/mL streptomycin sulfate (Sigma-Aldrich) and 100 U/mL penicillin (Sigma-Aldrich) in 10-cm culture dishes at 37 °C in 5% CO₂ humidified incubator for at least 24 h following established protocols. The adherent cells were discarded while the floating cells were cultured with macrophage colony-stimulating factor (M-CSF; R&D Systems) at 30 ng/mL for 48 h. To collect pure monocytes/macrophages, the cells are digested with Versene for ~4 min in 37 °C until most macrophages are de-associated. Then the cells are seeded in 24-well plate and cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL (Abcam, ab129136) for 5 days to induce the formation of mature osteoclasts. For treatments, 10⁻⁶ M MPS with/without 10⁻⁶ M RU486 is added to culture medium at designated timepoints and cultured for 24 h. TRAP activity is measured using a commercial kit (Sigma-Aldrich). Total RNA was extracted, and the mRNA level of ANG was measured by qRT-PCR.

Cultured media were collected and centrifuged at 1,200 g for 5 min. Media were supplemented with 17.5 μg mL⁻¹ phenyl-methylsulfonylfluoride, 1 μg mL⁻¹ pepstatin A, 2 μg mL⁻¹ aprotinin and 50 μg mL⁻¹ bacitracin, and stored at −80 °C. Human specific ELISA kits were used to measure angiopoietin-1, MCP-1, FGF-2, EGF, IGF-1, VEGF, VEGFR-1, PICP and IL-6 (all from R&D Systems), OPG (Bender MedSystems GmbH), RANKL (Biomedica Gruppe) and M-CSF (Abcam). For TGF-β1 detection, cell layers were washed with PBS and extracted with 5 × 10⁻² M Tris-HCl pH 8.0, 5 × 10⁻¹ M NaCl and 1% Triton X-100, supplemented with protease inhibitors as above described. The amount of TGF-β1 in the extracts was determined using a human specific ELISA (RayBiotech) and the data were normalized to the total protein amount in cell layers, as determined by a Bradford-based protein assay (Bio-Rad Laboratories Inc.).

CD14⁺ monocytes were isolated from human peripheral blood mononuclear cells (PBMCs, obtained from the Human Immunology Core, University of Pennsylvania) using the Human CD14 Positive Selection Kit (STEMCELL Technologies, Vancouver, Canada). After isolation, CD14⁺ cells were cultured in Minimum Essential Medium Alpha (MEMα) containing macrophage colony-stimulating factor (M-CSF; 20 ng/mL; Abcam) for 24 h. The cells were collected, seeded in 96-well plates (5 × 10⁴ cells/well) and cultured in MEMα containing M-CSF (20 ng/mL), receptor activator of nuclear factor kappa-Β ligand (RANKL; 40 ng/mL; R&D Systems, Minneapolis, MN, USA) and decitabine (0, 0.5, 1, 10 or 100 µM) for five days. Then, the cells were stained for TRAP and alkaline phosphatase (ALP) using the TRAPCP&ALP Double-stain Kit (Takara). TRAP-positive multinucleated cells (three or more nuclei) were counted as osteoclast-like cells.

In this study, proteins were extracted using a protein extraction kit (Sangon Biotech), and the bicinchoninic acid (BCA) assay (Sangon Biotech) was used to determine the total protein content. After denaturation for 5 min, total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA) via a constant current flow at 200 mA. Subsequently, the PVDF membranes were incubated with Bcl-2 (1:10,000), Bax (1:2000), Caspase-3 (1:5000), M-CSF (1:1000), RANKL (1:3000), OPG (1:3000), p-p65 (1:1000), p65 (1:1000), p-p38 (1:1000), p38 (1:5000), p-JNK (1:1000), JNK (1:1000), p-AKT (1:2000), AKT (1:2000), p-ERK (1:1000), ERK (1:1000), RANK (1:2000), NF-κB (1:1000), NFATc1 (1:10,000), and c-Fos (1:1000) antibodies (Abcam, Cambridge, MA) for 12 h at 4°C. TBS buffer was used to wash the PVDF membranes, and secondary antibodies (Abcam) were added and incubated at room temperature for 1 h. After the membranes were washed three times, chemiluminescent reagents were added, and the grayscale values of the bands were analyzed using ImageJ software. Each experiment was independently repeated 3 times. GAPDH was used to quantify the expression of various proteins.

Sorted primary CD115(+) precursors were cultured in α-minimal essential medium (MEM) containing 10% FBS, 1% penicillin-streptomycin solution and 50 ng ml^− 1 colony stimulating factor (M-CSF, Abcam). After 3 days, the medium was replaced with fresh medium. LA was added at a concentration of 100 μg/ml to stimulate the precursors. To inhibit the expression of Cadherin-11, the primary osteoclast precursors were transfected with Cadherin-11 siRNA using INVI DNA RNA Transfection reagent (Invigentech) for 24 h. The sequence of Cadherin-11 siRNA was CCAATGGACCAAGATTTAT. In some experiments, primary cells were treated with BYL719 (2.5 μM), GSK690693 (25 nM), 666–15 (50 nM), rapamycin (20 μM), or AMG-487 (30 μM).

Blood samples from all patients and controls were collected after overnight fast in plain tubes containing a separation gel. The blood samples were processed to collect serum and stored at −80°C until examination. Double-blinded quantitative measurement of VIP in serum was done by commercially available ELISA kit (Cusabio, Wu Han, China, Cat No. CSB-E08354h) based on the manufacturers’ instructions. The detection range was from 15.6 to 1000 pg/mL. According to the manufacturer, the intra-assay CV was < 8%, and the inter-assay CV was <10%. Serum TNF-α (Abcam, Cambridge, UK), IL-1β (Abcam), and M-CSF (Abcam) levels were also examined. All measurements were taken three times for each sample, and the results were averaged.